Structural Analysis of NAT Acetylation, Substrate Specificity and Polymorphisms

描述（由申请人提供）：芳基胺N-乙酰转移酶（NAT）可使作为烹饪、农药、烟草或染料副产品摄入的芳基胺膀胱致癌物解毒。它们通过将乙酰辅酶A的乙酰基转移到芳胺上来实现这一功能。与对照组相比，膀胱癌患者表现出芳胺加合物水平升高，其中一些已被证明是NAT的不良底物。作为这项建议的一部分，我们定义的NAT底物特异性的决定因素，揭示为什么某些芳胺致癌物是无法解毒。NAT是高度多态性的蛋白质，并且很大比例的群体具有体内催化活性降低的NAT变体。我们和其他人最近已经证明了这些变体通过泛素-蛋白酶体途径快速降解。我们的长期目标之一是使用NAT作为模型系统，以确定如何在细胞中识别泛素化靶点，并阐明导致其传递到蛋白酶体的机制。这项研究将为识别和消除异常蛋白质的质量控制途径提供基本信息。在以下三个一般领域开展工作。1)开发NAT底物特异性的通用模型。NMR和稳态动力学实验用于定义NAT底物特异性的决定因素，并且该知识用于产生可以乙酰化另外的芳胺致癌物的变体。2)确定导致NAT组成性泛素化的机制。在以前的工作中，我们证明了NAT组成性泛素化与其聚集状态有关，我们的初步数据表明这种泛素化发生在内质网。在拟议的研究中，我们确定NAT如何被识别为异常，其泛素化的决定因素，以及它们是否通过适用于其他聚集或错误折叠蛋白质的共同途径进行处理。3)定义乙酰化如何影响NAT结构和表面性质。这项研究的成功结果可以帮助预防膀胱癌，并为与错误折叠或聚集蛋白质相关的疾病（包括神经退行性疾病和癌症）带来新的治疗策略。这项研究与公共卫生的相关性这项研究对NAT相关的致癌作用具有治疗意义，特别是膀胱癌，以及与错误折叠或聚集蛋白相关的疾病，包括神经退行性疾病。NAT可以解毒被称为芳基胺的化学物质，基于人群的研究将NAT活性降低和逃避NAT解毒的芳基胺与膀胱癌联系起来。我们确定了某些芳胺如何逃避NAT解毒，以及为什么有些人的NAT蛋白在它们能够发挥蛋白质功能之前就被破坏了。

DESCRIPTION (provided by applicant): Arylamine N-acetyltransferases (NATs) detoxify arylamine bladder carcinogens that are ingested as cooking, pesticide, tobacco, or dye byproducts. They perform this function by transferring to the arylamine an acetyl group from acetyl-CoA. Bladder cancer patients exhibit elevated levels of arylamine-adducts compared to control groups, some of which have been demonstrated to be poor substrates for NATs. As part of this proposal, we define the determinants of NAT substrate specificity to reveal why certain arylamine carcinogens are unable to be detoxified. NATs are highly polymorphic proteins and a large percentage of the population harbor NAT variants with reduced catalytic activity in vivo. We, and others, have recently demonstrated such variants to be rapidly degraded through the ubiquitin-proteasome pathway. One of our long-term goals is to use NATs as a model system to determine how ubiquitylation targets are identified in cells and to elucidate the mechanism(s) that lead to their delivery to the proteasome. This research will provide fundamental information on quality control pathways that exist to recognize and eliminate aberrant proteins. The following three general areas are pursued. 1) Develop a general model for NAT substrate specificity. NMR and steady state kinetics experiments are used to define the determinants of NAT substrate specificity and this knowledge used to generate variants that can acetylate additional arylamine carcinogens. 2) Determine the mechanisms that lead to NAT constitutive ubiquitylation. In previous work, we demonstrated NAT constitutive ubiquitylation to be linked to its aggregation state and our preliminary data indicate such ubiquitylation to occur at the endoplasmic reticulum. In the proposed research, we determine how NATs are recognized as aberrant, the determinants of their ubiquitylation, and whether they are processed through a common pathway that applies to other aggregated or mis-folded proteins. 3) Define how acetylation affects NAT structure and surface properties. The successful outcome of this research could aid in the prevention of bladder cancer and lead to new therapeutical strategies for diseases associated with mis-folded or aggregated proteins, including neurodegenerative diseases and cancer. Relevance of this research to public health This research has therapeutical implications for NAT-associated carcinogenesis, especially bladder cancer, as well as diseases associated misfolded or aggregated proteins, including neurodegenerative diseases. NATs detoxify chemicals known as arylamines and population-based studies have connected reduced NAT activity and arylamines that evade NAT detoxification to bladder cancer. We determine how certain arylamines evade NAT detoxification and why the NAT proteins of some people are destroyed before they can perform their protein function.